An inhibitor of TRPV1 channels isolated from funnel web spider venom
An inhibitor of TRPV1 channels isolated from funnel web spider venom
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DOI：
10.1021/bi051494l
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发表时间：
2005-11-29
期刊：
影响因子：
2.9
通讯作者：
Swartz, KJ
中科院分区：
文献类型：
--
作者：
Kitaguchi, T;Swartz, KJ
辣椒素受体通道 (TRPV1) 是非选择性阳离子通道，可整合感觉神经元中的多种有害刺激。为了鉴定这些通道的新抑制剂，我们筛选了毒液库中针对 TRPV1 通道的活性，并发现来自北美漏斗网蜘蛛 Agelenopsis aperta 的毒液具有强大的抑制活性。使用反相 HPLC 对毒液进行分级，纯化出两种酰基多胺毒素 AG489 和 AG505，它们从膜的细胞外侧抑制 TRPV1 通道。进一步表征了 AG489 的活性，发现该毒素可抑制 TRPV1 通道，在 -40 mV 下的 K-i 为 0.3 μM。 AG489 对 TRPV1 通道的抑制具有强烈的电压依赖性，在正电压下抑制会减轻，这与毒素通过孔阻塞机制抑制通道一致。我们在整个 TM5-TM6 连接器（被认为形成 TRPV1 通道的外孔的区域）中使用扫描诱变来识别改变毒素亲和力的孔突变。四种突变体显着降低了毒素亲和力，几种突变体增加了毒素亲和力，这与 TM5-TM6 连接子形成 TRPV1 通道的外前庭以及 AG489 是孔阻断剂的观点一致。
Capsaicin receptor channels (TRPV1) are nonselective cation channels that integrate multiple noxious stimuli in sensory neurons. In an effort to identify new inhibitors of these channels we screened a venom library for activity against TRPV1 channels and found robust inhibitory activity in venom from Agelenopsis aperta, a north American funnel web spider. Fractionation of the venom using reversed-phase HPLC resulted in the purification of two acylpolyamine toxins, AG489 and AG505, which inhibit TRPV1 channels from the extracellular side of the membrane. The activity of AG489 was characterized further, and the toxin was found to inhibit TRPV1 channels with a K-i of 0.3 mu M at -40 mV. Inhibition of TRPV1 channels by AG489 is strongly voltage-dependent, with relief of inhibition at positive voltages, consistent with the toxin inhibiting the channel through a pore-blocking mechanism. We used scanning mutagenesis throughout the TM5-TM6 linker, a region thought to form the outer pore of TRPV1 channels, to identify pore mutations that alter toxin affinity. Four mutants dramatically decrease toxin affinity and several mutants increase toxin affinity, consistent with the notion that the TM5-TM6 linker forms the outer vestibule of TRPV1 channels and that AG489 is a pore blocker.